Cancer is generally a disease of the intracellular signaling system. Normal cells respond to many extracellular signals by proliferating, differentiating or otherwise altering their metabolic activity. Such signals are received at the cell surface and converted by a system of signal transduction proteins into a message decipherable by the cell. The message is responsible for subsequent regulation of cell processes.
An example of proteins that are involved in the signal transduction pathway are the mitogen-activated protein (MAP) kinases. The MAP kinases are believed to be directly involved in the regulation of genes that are responsible for cell proliferation.
The MAP kinase superfamily of genes includes three families. One of these families includes the genes that encode ERK-1 and ERK-2. The second family includes the genes that encode the stress activated protein kinases, such as JUN kinase The third family includes the gene that encodes p38.
The MAP kinases, in turn, are regulated by various levels of upstream regulatory proteins. The mechanism of regulation of MAP kinases is reversible protein phosphorylation.
The first level of upstream MAP kinase regulatory proteins includes the family of MAP kinase kinases, such as MEK. The MAP kinase kinases, in turn, are regulated by the second level of regulatory proteins, the MAP kinase kinase kinases.
It is believed that cancer is commonly caused by defects in the genes responsible for signal transduction. Such defective genes are called oncogenes. Oncogenes can lead to the overexpression of one or more signal transduction proteins causing the cell nucleus to receive an inappropriate signal to proliferate. Defective signals can occur through a variety of mechanisms.
The proteins expressed by oncogenes, called oncoproteins, typically act directly as transactivators and regulators of the synthesis of RNA and DNA. Many oncogenes are members of the family of MAP kinase kinases and MAP kinase kinase kinases. Some examples of oncogenes that have been widely studied are ras, raf-1, myc, ski, myb, fos and jun. See Blenis, J. "Signal Transduction Via MAP Kinase: Proceed at Your Own Risk," Proc. Natl. Acad. Sci. U.S.A., Vol. 90, 5889-5892 (1994); Cobb, et al., "How MAP Kinases Are Regulated," J. Biol. Chem., Vol. 270, 14843-14846 (1995), and Janes, et al., "Activation of the Ras Pathway In Breast Cancer Cells," Oncogene, Vol. 9, 3601-3608 (1994).
For example, constitutively active MAP kinases are believed to induce oncogenicity when a MAP kinase kinase (MEK) is expressed in fibroblasts. Brunet e al., "MAP Kinase Module: Role in the Control of Cell Proliferation," Comptes. Rendus. Sci. Soc. Biol., Vol. 189, 43-57 (1995); Brunet et al., "Constitutively Active Mutants of MAP Kinase Induced Growth Factor-Relaxation and Oncogenicity When Expressed in Fibroblasts," Oncogene, Vol. 9, 3379-3387 (1994).
A number of viral and cellular genes have been identified as potential oncogenes. The products of oncogenes are classified according to their cellular location, for example, secreted, surface, cytoplasmic, and nuclear oncoproteins.
The products of nuclear oncogenes have the ability to induce alterations in gene regulation leading to abnormal cell growth and ultimately neoplasia. As a result of the expressed products of oncogenes being involved in the formation of potentially malignant neoplastic cell growth, there has been much focus on methods of inhibiting oncoprotein expression.
A technique that is becoming prevalent to inhibit expression of a target protein, such as an oncoprotein, is the use of antisense oligonucleotides. Antisense oligonucleotide inhibition of oncogenes has proven to be a useful tool in understanding the roles of the various oncogene families.
Antisense oligonucleotides are small oligonucleotides that are complementary to, and thus able to specifically hybridize with, the mRNA transcript of the target gene. In some instances, the antisense oligonucleotides bind to the major groove of the double stranded (ds) DNA that encodes the target protein to form a triple helix or antigene. Binding to either the mRNA or the dsDNA inhibits expression of the targeted protein. A discussion of such triple helixes is found in Stull et al., "Antigene, Ribozyme and Aptamer Nucleic Acid Drugs: Progress and Prospects," Pharm. Res., Vol. 14, No. 4, 465-483 (1995), incorporated herein by reference.
Considerable attention has been directed to how the expressed products of oncogenes alter the signal transduction pathway of cells. Currently, the focus on signal transduction pathway alterations is primarily directed to the upstream regulators of MAP kinases, such as ras, raf-1, and MEK. Some examples of this approach can be found in U.S. Pat. No. 5,582,986, which discloses antisense oligonucleotides for inhibition of the ras gene, U.S. Pat. No. 5,597,719, which discloses human 14-3-3 proteins that modulate raf-1 activity, and U.S. Pat. No. 5,525,625, which discloses flavone compounds that inhibit the activity of MEK.
In addition, Holt et al., Mol. Cell Biol., Vol. 8, 963-973 (1988), have shown that antisense oligonucleotides hybridizing specifically with mRNA transcripts of the oncogene c-myc, when added to cultured HL60 leukemic cells, inhibit proliferation and induce differentiation. Anfossi et al., Proc. Natl. Acad. Sci., Vol. 86, 3379-3383 (1989), have shown that antisense oligonucleotides specifically hybridizing with mRNA transcripts of the c-myb oncogene inhibit proliferation of human myeloid leukemia cell lines. Wickstrom et al., Proc. Nat. Acad. Sci., Vol. 85, 1028-1032 (1988), have shown that expression of the protein product of the c-myc oncogene as well as proliferation of HL60 cultured leukemic cells are inhibited by antisense oligonucleotides hybridizing specifically with c-myc mRNA.
However, these strategies of inhibiting or inactivating the upstream regulators of MAP kinase, such as ras, raf-1 and MEK, have generally not been effective. It is becoming apparent that the proliferation pathway blocked by the inhibition may be replaced by other pathways that promote unregulated cell proliferation. The replacement pathway may occur in the malignant cells treated with the antisense oligonucleotides, or in clones of other malignant cells that co-exist with the treated cells.
In addition to the inhibition of the upstream regulators of MAP kinase discussed above, the inhibition of expression of MAP kinase itself has been demonstrated in vitro and in vivo. Such inhibition has, to date, been used mainly as a general research tool.
For example, Sale et al., disclose the use of antisense oligonucleotides to investigate the role of MAP kinase in the differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis," Sale et al., EMBO J., Vol. 14, No. 4, 674-684 (1995). Gao et al., disclose the use of the antisense oligonucleotides of Sale et al., to investigate the role of MAP kinase in F9 teratocarcinoma stem cell progression. Gao et al., J. Biol. Chem., Vol. 271, No. 15, 9002-9008 (1996).
The role of MAP kinases in cancer has been investigated. These findings however, have been inconclusive, and have not provided new routes of inhibiting malignant neoplastic cell growth.
For example, it has been reported that the activity of MAP kinases ERK-1 and ERK-2 can be correlated to the overexpression of elF-4E in CREF cells and the malignancy of each cell line. Graff, Jeremy R., "Messenger on a Translation and to Malignancy," The mRNA Cap-Binding Protein, elF-4E, as an Integral Component of Malignancy in Cloned Rat, Dissertation submitted to University of Kentucky, Chapter 6, 110-130 (1994). However, no hypothesis as to the role of MAP kinase in cancer was made. Moreover, studies that illustrate the hyperactivation of MAP kinase do not provide any suggestions as to the cause of the hyperactivation. These findings do not provide new routes of treating malignant neoplastic cell growth, such as primary breast carcinoma.
Likewise, there has been a report of increased MAP kinase expression in cultured non-small cell lung carcinomas and breast cancer cell lines. However, only about a third of the breast cancer cell lines examined exhibited changes in MAP kinase expression. An inconsistent pattern was observed in which some cell lines exhibited more ERK-1 and others exhibited more ERK-2. Cobb, Melanie H., "The Role of MAP Kinase Pathway in Breast Cancer," National Technical Information Service, Accession No. AD-8301 655/7/XAB (1995). As a result of the se inconsistent findings, the author stated that she was unable to make even an initial hypothesis as to the role of MAP kinase in cancer. Another difficulty with this study, and other studies with cultured cell fines, is that one cannot extrapolate the results with the results obtained with non-cultured cells from biopsies.
At the present time, treatment of cancer primarily relies on the use of radiation and/or chemotherapeutic agents, such as vinblastine or adriamycin. However, it is widely recognized that the side effects of such treatments are at times severe, making these treatment strategies very unpopular.
Another problem related to cancer is the reliance on gross pathology to make an initial prognosis of malignant neoplastic cell growth. In many malignant neoplastic cell growths, early detection is difficult unless there is a phenotypical alteration to indicate malignancy. While there has been some progess in the development of assays for the detection of cancer in the early stages (e.g., prostate and melanoma), such assays are not applicable to other cancers, such as breast cancer.
In view of the above, it is apparent that there is a continuing need in the art for an effective treatment for cancer that removes the necessity of the administration of radiation and chemotherapeutic agents. Likewise, there is a continuing need in the art for a method of identifying and monitoring potentially malignant neoplastic growths which would allow for early detection and staging of the malignancy.
Accordingly, it is the object of the present invention to provide more effective methods that overcome the disadvantages of the prior art methods for identifying and monitoring potentially malignant neoplastic cell growth, and for treating cancer.